Identification and capsular serotype sequetyping of Streptococcus pneumoniae strains

Abstract

Correct identification of Streptococcus pneumoniae (pneumococcus) and differentiation from the closely related species of the Mitis group of the genus Streptococcus, as well as serotype identification, is important for monitoring disease epidemiology and assessing the impacts of pneumococcal vaccines. In this study, we assessed the taxonomic identifications of 422 publicly available genome sequences of S. pneumoniae, S. pseudopneumoniae and S. mitis, using different methods. Identification of S. pneumoniae, by comparative analysis of the groEL partial sequence, was possible and accurate, whereas S. pseudopneumoniae and S. mitis could be misclassified as S. pneumoniae, suggesting that groEL is unreliable as a biomarker for differentiating S. pneumoniae from its closest related species. The genome sequences of S. pneumoniae and S. pseudopneumoniae fulfilled the suggested thresholds of average nucleotide identity (ANI), i.e., > 95% genome sequence similarity to the sequence of respective type strains for identification of species, whereas none of the S. mitis genome sequences fulfilled this criterion. However, ANI analyses of all sequences versus all sequences allowed discrimination of the different species by clustering, with respect to species type strains. The in silico DNA-DNA distance method was also inconclusive for identification of S. mitis genome sequences, whereas presence of the \"Xisco\" gene proved to be a reliable biomarker for S. pneumoniae identification. Furthermore, we present an improved sequetyping protocol including two newly-designed internal sequencing primers with two PCRs, as well as an improved workflow for differentiation of serogroup 6 types. The proposed sequetyping protocol generates a more specific product by generating the whole gene PCR-product for sequencing, which increases the resolution for identification of serotypes. Validations of both protocols were performed with publicly available S. pneumoniae genome sequences, reference strains at the Culture Collection University of Gothenburg (CCUG), as well as with clinical isolates. The results were compared with serotype identifications, using real-time Q-PCR analysis, as well as the Quellung reaction or antiserum panel gel-precipitation. Our protocols provide a reliable diagnostic tool for taxonomic identification as well as serotype identification of S. pneumoniae.